Ryanodine binding sites measured in small skeletal muscle biopsies

• Published in: Scandinavian journal of clinical and laboratory investigation Vol. 57; no. 7; pp. 569 - 580
• Main Authors: Lunde, P. K., Sejersted, O. M.
• Format: Journal Article
• Language: English
• Published: Oslo Informa UK Ltd 1997; Taylor & Francis; Scandinavian University Press
• Subjects: Adenosine Triphosphate - pharmacology; Animals; Binding, Competitive - drug effects; Binding, Competitive - physiology; Biological and medical sciences; Biopsy, Needle; Calcium - pharmacology; Cholic Acids; Cryopreservation; Detergents; EDL; Egtazic Acid - analogs & derivatives; Fundamental and applied biological sciences. Psychology; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; Magnesium - pharmacology; muscle biopsies; Muscle, Skeletal - chemistry; Muscle, Skeletal - pathology; Octoxynol; Poloxalene; Potassium Chloride - pharmacology; Rats; Rats, Wistar; ryanodine receptor; Ryanodine Receptor Calcium Release Channel - analysis; Ryanodine Receptor Calcium Release Channel - metabolism; Sarcoplasmic Reticulum - chemistry; Soleus; Striated muscle. Tendons; Time Factors; Tritium; Vertebrates: osteoarticular system, musculoskeletal system; Human; Ryanodine; Vertebrata; Mammalia; Calcium ion; Rat; Biopsy; Rodentia; Ionic channel; Binding site; Striated muscle; Quantitative analysis
• ISSN: 0036-5513; 1502-7686
• DOI: 10.3109/00365519709055279

A method allowing measurement of the concentration of [3H]ryanodine binding sites in small skeletal muscle specimens (<10-20 mg) was developed. A membrane fraction containing 87% of the [3H]ryanodine binding sites of the tissue and exhibiting one single KD of 18-27 nmol I-1 in rat and 8 nmol I-1 in human muscles (p < 0.05) was obtained. Maximum binding to rat EDL and soleus muscles equalled 59.1 and 16.2 pmol g-1 wet wt, whereas in human gluteus muscles binding was 12.3 pmol g-1 wet wt. The [3H]ryanodine binding showed a dependency on Mg2+ and pH similar to previously published results. As measured by Ca2+ selective mini-electrodes, the [Ca2+] causing 50% of maximum [3H]ryanodine binding (K0.5) was 200-400 nmol I-1 for different muscles. [Ca2+] higher than 1 mmol I-1 caused strong inhibition of the [3H]ryanodine binding, and both high and low [Ca2+] caused rapid dissociation of the complex. At ionic strength lower than 100 mmol I-1, more than 50% of the [3H]ryanodine was bound to particles with size less than 1.2 μn which were not retained by GF/C filters. Thus, we have obtained an almost complete quantitative recovery of functional RyRs from small muscle specimens exhibiting high affinity for Ca2+, which stimulated ligand binding.